Abstract
The low density lipoprotein receptor-related protein-1 (LRP1) is known to serve as a chylomicron remnant receptor in the liver responsible for the binding and plasma clearance of apolipoprotein E-containing lipoproteins. Previous in vitro studies have provided evidence to suggest that LRP1 expression may also influence high density lipoprotein (HDL) metabolism. The current study showed that liver-specific LRP1 knock-out (hLrp1(-/-)) mice displayed lower fasting plasma HDL cholesterol levels when compared with hLrp1(+/+) mice. Lecithin:cholesterol acyl transferase and hepatic lipase activities in plasma of hLrp1(-/-) mice were comparable with those observed in hLrp1(+/+) mice, indicating that hepatic LRP1 inactivation does not influence plasma HDL remodeling. Plasma clearance of HDL particles and HDL-associated cholesteryl esters was also similar between hLrp1(+/+) and hLrp1(-/-) mice. In contrast, HDL secretion from primary hepatocytes isolated from hLrp1(-/-) mice was significantly reduced when compared with that observed with hLrp1(+/+) hepatocytes. Biotinylation of cell surface proteins revealed decreased surface localization of the ATP-binding cassette, subfamily A, member 1 (ABCA1) protein, but total cellular ABCA1 level was not changed in hLrp1(-/-) hepatocytes. Finally, hLrp1(-/-) hepatocytes displayed reduced binding capacity for extracellular cathepsin D, resulting in lower intracellular cathepsin D content and impairment of prosaposin activation, a process that is required for membrane translocation of ABCA1 to facilitate cholesterol efflux and HDL secretion. Taken together, these results documented that hepatic LRP1 participates in cellular activation of lysosomal enzymes and through this mechanism, indirectly modulates the production and plasma levels of HDL.
Highlights
It is important to note that these studies on the influence of hepatic lipoprotein receptor-related protein-1 (LRP1) on lipoprotein levels and atherosclerosis were performed with hypercholesterolemic mice that lacked low density lipoprotein (LDL) receptor and apolipoprotein E (apoE)
Hepatocyte Glycosphingolipid Quantification—Primary hepatocytes from hLrp1ϩ/ϩ and hLrp1Ϫ/Ϫ mice cultured at a density of 2.0 ϫ 106 cells/well in 10-cm cluster dishes were washed three times in phosphate-buffered saline (PBS) and scraped in lysis buffer consisting of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 0.25 M sucrose
Similar elevation of prosaposin and decreased cathepsin D levels were observed in freshly isolated liver of hLrp1Ϫ/Ϫ mice (Fig. 6C). These results suggest that decreased intracellular levels of the lysosomal enzyme cathepsin D along with the accompanying defect in prosaposin activation are responsible for reduced cell surface ABCA1 and decreased high density lipoprotein (HDL) production in hLrp1Ϫ/Ϫ hepatocytes
Summary
Animals and Diets—Animals were maintained in a pathogen-free facility, and all experimental procedures were performed with protocols approved by the University of Cincinnati Institutional Animal Use and Care Committee. Cell Surface Labeling and Immunoblotting—Primary hepatocytes plated in 100-mm tissue culture dishes (2 ϫ 106 cells/dish) were washed five times in ice-cold phosphate-buffered saline and incubated twice for 20 min at 4 °C with PBS containing 0.5 mg/ml sulfo-NHS-SS-biotin (Pierce) [6]. Following three washes in PBS, cell surface protein-biotin complexes were eluted from the streptavidin beads with 4ϫ Tris-glycine gel loading buffer containing 10% (v/v) -mercaptoethanol at 37 °C for 30 min. Hepatocyte Glycosphingolipid Quantification—Primary hepatocytes from hLrp1ϩ/ϩ and hLrp1Ϫ/Ϫ mice cultured at a density of 2.0 ϫ 106 cells/well in 10-cm cluster dishes were washed three times in PBS and scraped in lysis buffer consisting of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 0.25 M sucrose. A value of p Ͻ 0.05 was considered to be statistically significant
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